INTRODUCTION environmental related stress in fish (Afonso et al

INTRODUCTION

 

          The
River Warri is an important river in the Niger Delta of southern Nigeria. The
economic importance of the river is predicated on its rich biota (Egborge 1986;
Egborge and Tawarri 1987; Opute 1991, Tetsola and Egborge 1991; Ikomi 1995)
that provide various kinds of fishes for human consumption, means of inland
water transport for most communities in the region and the site of a large port
at Forcados (Ikomi 2000). However, the river is highly polluted due to the
presence of oil / petrochemical complex (Egborge 1991), enormous oil
exploration activities in Warri and its environs and discharge of domestic and
other industrial effluent into the river (Egborge, 1991).

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          The
extent of pollution of Warri Rivers has been monitored by an array of the
scientific communities (Atuma and Egborge, 1986 Egborge 1991; 1994)culminating
in changes in water quality indices (Egborge and Benka – Coker, 1986; Ikomi
1993; 2000) and bioconcentratin of trace metals in fish (Kakulu et al 1997;
Ezemonye 1992; Agada, 1994).

          Fish
in natural environment are often exposed to a variety of stressors that can
adversely affect their health. Thus, there is a need to develop tools to assess
environmental related stress in fish (Afonso et al 2003). Some of the
biondicators of stress in fish include increase in levels of plasma cortisol,
glucose and lactate (Barton and Iwama, 1991); induction of heat shock protein
(Iwama et al 1999); glucose and drug metabolizing enzymes (Isamah and Asagba,
2004).

          The
induction of oxidative stress in fish by polluted environment is well
documented (Bainy et al 1996; DiGuilio et al 1989; Hai 1997). This study
reports on oxidative stress on Clarias heterobranchus from Warri River
in southern Nigeria.

Materials and Method

Study Area

          The
study area is Warri
River. It is located
between latitude 50211-60.001N and
longitude 50251E. The river took its source at Utagba –
Uno and flow towards south west through Eziokpor, Amai, Otorho – Abraka, Warri
and emptying into the sea at Forcados (Tetsola and Egborge, 1991).

            The
fishes, Clarias heterobranchus were obtained from the upper, middle and near
the lower course of the Warri
River between August and
September, 2004. The fishes were caught using instrument made locally. They
were transported alive to the laboratory and allowed to stabilize for one week before
they were dissected to extract organs and tissues of interest. Similar fish of comparable size
were obtained from a commercial fish pond located in Abraka and were used
as control.
The fishes were sorted and duly identified by the Department of Zoology, Delta State
University, Abraka, Nigeria.

          A
total of 30 samples of mature size-matched male C heterobranchus (average wet
weight 340+5g and average wet length 38.7+3.2cm) were collected
from each site. Males were chosen for this study in order to be sex specific
since biomarkers have been found to be sex related (Afonso et al 2003). All the
reagents used were of analytical grade.

 

 

METHODS

PREPARATION
OF EXTRACT FOR THE DETERMINATION OF LIPID PEROXIDATION

          Of
the isolated organs 0.5g were separated and homogenized with 10ml of ice-cold
0.05M phosphate butter pH 7.0 containing 1% (w/v) Triton X-100, excess
butylated hydroxyl toluene (BHT) and a few crystals of protease inhibitor,
phenylmethylsulfonyl fluoride using an MSE blender immersed in ice. Triton
X-100 solubilizes membrane-enclosed organells while BHT prevents in vitro
oxidation of lipid during homogenization. The extract was centrifuged at 7000g
for 20 min (40C). The supernatant (S1) was used for the
determination of lipid peroxidation by the method of Hunter et al (1963) as
modified by Gutteridge and Wilkins (1982).

EXTRACTION
AND ASSAY OF CATALASE

          Catalase
was measured with a similar (S1) fraction after addition of 1% (V/V)
of ethanol and incubation at 40C for 15 min. This treatment is
reported to reverse the inactivation of catalase, which takes place, by the
formation of compound 11 (Cohen et al, 1970). Catalase activity was determined
according to Beers and Sizer (1952) by measuring the decrease in the H202
concentration, at an absorbance of 240nm. An extinction coefficient for H202
of 40M-1 cm-1 (Abei, 1974) was used in the calculation.

 

EXTRACTION AND ASSAY OF SUPEROXIDE
DISMUTASE

          An
aliquot of the supernatant (S1) was precipitated on ice with 0.30
volume of chloroform/ methanol (3:5v/v) stirred for 20min and centrifuged at
7000g at 40C. The obtained supernatant (S2) was used for
the assay of superoxide dismutase (SOD) activity, which was based on its
ability to inhibit the oxidation of epinephrine by superoxide anion (Aksnes and
Njaa, 1981). One unit of superoxide dismutase activity is defined as the amount
of enzyme required for 50% inhibition of the oxidation of epinephrine to
adrenochrome at 480nm per min (Misra and Fridovich, 1972). Manganese dependent
SOD was analyzed in the presence of 1mM NaCN to suppress Cu-ZnSOD activity and
the cytosolic Cu-ZnSOD activity was determined as the difference between total
and cyanide – sensitive enzyme activity (Crapo et al, 1978). The enzyme
activities were assayed with an SP 1800 UV/VIS Spectrophotometer.

STATISTICAL ANALYSIS

The mean and standard
error of the mean (SE), for the various parameters, were compared for
statistical significant difference using the students –T test. P. values

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